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Ann. N.Y. Acad. Sci. 1050: 243–249 (2005). © 2005 New York Academy of Sciences.doi: 10.1196/annals.1313.025

A Deiminated Viral Peptide to Detect Antibodies in Rheumatoid Arthritis

GIUSEPPINA MERLINI, CONSUELO ANZILOTTI, DANIELE CHIMENTI, CRISTINA TOMMASI, STEFANO BOMBARDIERI, AND PAOLA MIGLIORINI

Clinical Immunology Unit, Department of Internal Medicine, University of Pisa, Pisa, Italy

ABSTRACT: The data presented suggest that a deiminated viral peptide isspecifically recognized by antibodies contained in rheumatoid arthritis (RA)sera. Antipeptide antibodies are not associated with the presence or severity ofspecific manifestations of RA, but are more frequent in subjects with erosivearthritis. Taking into account the association with rheumatoid factor and witherosive arthritis, we can conclude that antipeptide antibodies are markers ofsevere forms of RA. Our data also show familial aggregation of anticitrullinatedpeptide antibodies.

KEYWORDS: rheumatoid arthritis (RA); antipeptide antibodies; deiminatedviral peptide

Rheumatoid arthritis (RA) is the most common autoimmune disease in humans. It ischaracterized by the inflammation of synovial membranes, which can lead to thedestruction of synovial joints. RA is diagnosed primarily on clinical manifestations.Serological supporting evidence has long been restricted to the determination of IgMrheumatoid factor; this antibody, however, occurs frequently in many inflammatorydiseases and in healthy elderly individuals. In 1964, two Dutch scientists first dis-covered autoantibodies (antiperinuclear factor, APF) that label a component of thekeratohyalin granules surrounding the nucleus of differentiating human buccalmucosa cells. These autoantibodies can be found with a frequency of 49–91% in RApatients’ sera, and their specificity for the disease is 73–79%.1

In 1979, it was shown that sera from many RA patients bound the stratum corneumof rat esophagus (antikeratin antibodies, AKA). AKA are detected in 36–59% of RAsera and are present almost exclusively in this disease (specificity: 88–99%).2

APF and AKA are detected by indirect immunofluorescence. Despite their goodsensitivity and high specificity, they have never been routinely used: standardization,in fact, is difficult, and the reproducibility is scarce. In addition, not every normalhealthy donor has buccal mucosa cells suitable for APF detection.

The targets of AKA in rat esophagus epithelium are proteins of heterogeneouscharge and different molecular weight.3 In human epidermis, AKA recognize a 40-kDa protein that has been identified as a neutral/acidic isoform of filaggrin.4 Filaggrin

Address for correspondence: Paola Migliorini, M.D., Clinical Immunology Unit, Department ofInternal Medicine, Via Roma 67, 56126 Pisa, Italy. Voice: +39-50-558609; fax: +39-50-558630.

p.migliorini@med.unipi.it

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is a cytokeratin filament–aggregating protein synthesized as a high molecular weightprecursor named profilaggrin and stored in keratohyalin granules. During epidermaldifferentiation, profilaggrin is cleaved, dephosphorylated, and deiminated: theenzyme peptidylarginine deiminase (PAD) transforms, in fact, a variable number ofarginine residues into citrulline, thus generating proteins of heterogeneous and moreacidic charge. Both APF and AKA recognize the neutral/acidic forms of filaggrin inbuccal and esophageal mucosa cells. Thus, two RA-associated autoantibodiespreviously considered different actually recognize the same target,5 and both can belabeled antifilaggrin antibodies (AFA).

The posttranslational modification of arginine residues is necessary to generateepitopes recognized by AFA. In fact, recombinant filaggrin is not bound by RA serabefore in vitro treatment with PAD,6 and only filaggrin sequences containingcitrulline instead of arginine detect RA-associated antibodies.7

Immunoenzymatic assays (enzyme-linked immunosorbent assay, ELISA) basedon such sequences have been developed7 and used to screen patient sera. A moresensitive assay is obtained, with a modified peptide that optimally exposes thecitrulline moiety and thus allows the detection of antibodies in up to 70% RApatients.8

A comparative evaluation of sequences recognized by AFA shows that theircritical feature is the presence of citrulline flanked by neutral amino acids such asglycine, serine, or threonine.6 Similar amino acid repeats are commonly found in viralproteins.7

One of the nuclear proteins encoded by Epstein-Barr virus, EBNA I, contains asequence (35–58) in its N-terminal region that bears a strong sequence homology tothe C-terminal protein of SmD, the spliceosome protein recognized by autoantibodiesin lupus sera.9,10 We synthesized this sequence by substituting arginine with citrullineand used it to test sera from patients with connective tissue disorders. The results weobtained indicate that only sera from RA patients react with a citrulline-containingviral peptide.

PATIENTS AND METHODS

Patients

Sera were obtained from 170 RA patients and 147 control subjects, including 20mixed cryoglobulinemia (MC) patients, 20 systemic lupus erythematosus (SLE) pa-tients, 30 systemic sclerosis (SSc) patients, and 77 normal healthy subjects (NHS). Adiagnosis of RA, SLE, and SSc was based on the ACR criteria;11–13 MC was diag-nosed in the presence of Meltzer’s triad (purpura, weakness, and arthritis/arthralgia)and of cryoglobulins in the sera.

Anticitrullinated Peptide Antibodies Detection

We developed an ELISA to detect anticitrullinated peptide antibodies. Briefly,ELISA plates (Nunc) were coated with a synthetic peptide (corresponding to theamino acid sequence 35–58 of the EBNA I protein) in which all the arginine residuesare substituted with citrulline diluted at 5 µg/mL in phosphate buffer solution (PBS).

245MERLINI et al.: A DEIMINATED VIRAL PEPTIDE

After blocking with PBS 3% bovine serum albumin (BSA), sera diluted 1/200 inPBS, 1% BSA, and 0.05% Tween were incubated for 3 h at room temperature. Afterrepeated washings, anti-human IgG F(ab′)2 fragment labeled with alkaline phos-phatase (Sigma Chemical Co., St. Louis, MO) diluted 1:3000 in PBS, 1% BSA, and0.05% Tween-20 was added to the wells and incubated for 3 h at room temperature.After washings, the bound enzyme activity was measured using para-nitrophenyl-phosphate as substrate (Sigma Chemical Co.).

Results are expressed as the percentage of an internal positive sample; positivitythreshold of the test is 27% (higher than the 97.5th percentile found in a group ofhealthy control subjects).

Statistical Analysis

All the variables were analyzed independently using the Fisher’s exact test forcontingency tables. A P value of <.05 was considered significant.

RESULTS

We studied 170 serum samples from RA patients (121 women and 49 men; meanage: 60 years; range: 20–88 years; mean disease duration: 8 years; range: 6 months to41 years) and from 147 control subjects (77 blood donors, 20 MC, 20 SLE, 30 SSc).

Among RA patients, 78/170 (44%) were positive for antipeptide antibodies. Inthese patients, we evaluated the presence of xerostomia, xerophthalmia, peripheralvasculitis, rheumatoid nodules, morning stiffness, symmetric arthritis, active arthritis,erosive arthritis, rheumatoid factor, and levels of C reactive protein.

The presence of antipeptide antibodies was associated with positivity forrheumatoid factor (P < .0001) and erosive arthritis (P < .05).

We also analyzed antipeptide antibodies in familial cases of RA. Sera from 16families in which 2 or more siblings were affected by the disease were studied. In12/16 families, the levels of antipeptide antibodies were similar in affected siblings;they were discordant in only 4 families.

DISCUSSION

The data presented in this paper suggest that a deiminated viral peptide is specif-ically recognized by antibodies contained in RA sera.14 In fact, IgG antibodies thatbind the deiminated EBNA I 35–58 sequence are detectable in 44% of RA sera. Onthe contrary, antibodies with such a specificity are not present in normal subjects orin other connective tissue disorders15 (FIG. 1). These antibodies are more frequentlydetected in rheumatoid factor–positive patients, but this association is not due to theinterference of rheumatoid factor with their detection. In fact, MC sera that containrheumatoid factor at high titers do not react with the viral peptide.

Antipeptide antibodies are not associated with the presence or severity of specificmanifestations of RA, but are more frequent in subjects with erosive arthritis. Thus,taking into account the association with rheumatoid factor and with erosive arthritis,we can conclude that antipeptide antibodies are markers of severe forms of RA.

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Our data also show familial aggregation of anticitrullinated peptide antibodies(FIG. 2). In familial cases of RA, the affected siblings are in most cases both positiveor both negative for antipeptide antibodies. Unfortunately, in only one family, a non-affected sibling also was tested; thus, we cannot discriminate between shared genesand shared environment. Familial aggregation has been demonstrated for differentfamilies of autoantibodies, such as antinuclear, antiphospholipid,16 antigoblet,17 andantithyroid.18

In most cases, autoantibodies have been analyzed in relatives and spouses ofsubjects with autoimmune disorders.16,17 In the case of antithyroid antibodies, theprevalence of antiperoxidase, antithyroglobulin, and anti–thyroid-stimulating hor-mone receptor antibodies has been analyzed in twin siblings affected by autoimmunethyroid diseases and healthy twins matched for age, sex, and zygosity.

Significantly more monozygotic twin siblings were positive for two or more auto-antibodies than dizygotic twin siblings, strongly supporting the hypothesis that thefamilial aggregation of thyroid autoantibodies is genetically determined. The geneticcontrol of the production of anticitrullinated peptide antibodies may be exerted atmultiple levels: T and B receptor repertoire, antigen processing, and presentation. Inthis respect, it is of interest that citrullinated peptides can be bound by the RA-associated DR4 alleles more efficiently than the corresponding noncitrullinatedpeptides. The in vivo relevance of this observation is indicated by the specific T-cellresponse elicited by citrullinated peptides in HLA-DR4 transgenic mice.19

Citrullination is a posttranslational modification operated by PAD and deeply in-fluenced by tissue expression and activity of these enzymes. Genetic factors regulating

FIGURE 1. Anticitrullinated viral peptide antibodies in connective tissue diseases. Thegraph shows the reactivity with citrullinated peptide measured in sera from patients with RAand control subjects (other connective tissue diseases and healthy donors). The reactivity isexpressed as a percentage of a reference serum. The dashed line represents the cutoff value.RA, rheumatoid arthritis; MC, mixed cryoglobulinemia; SLE, systemic lupus erythematosus;SSc, systemic sclerosis; NHS, normal healthy subjects.

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PAD activity have been analyzed recently in subjects affected by RA. In a Japanesepopulation, it has been shown that a PADI4 haplotype is associated with highermRNA stability and with susceptibility to RA.20 These data have not been confirmedin Caucasian populations,21,22 suggesting that further studies are needed to unravelthe complex relationship between PAD genes, deimination, and RA.

In conclusion, the anticitrullinated peptide antibodies we described are detectableexclusively in RA and are associated with severe forms of the disease. Both featuresare typical of AFA, suggesting the overlap of the two antibody populations. How-ever, filaggrin is not the only deiminated protein presently known, and it is not ex-pressed in synovia. Other deiminated proteins have been detected in synovial tissueand one, fibrin, is also a target of RA-specific antibodies.23

Therefore, AFA should be more comprehensively labeled as anticitrullinated pep-tide antibodies (ACPA).24,25 Because any protein containing several arginine resi-dues in the right amino acidic context can be deiminated by PAD becoming apotential target of ACPA, our deiminated viral peptide might be considered one ofthe substrates to detect ACPA.

REFERENCES

1. NIENHUIS, R.L.F. & E.A. MANDEMA. 1964. A new serum factor in patients with rheu-matoid arthritis: the antiperinuclear factor. Ann. Rheum. Dis. 23: 302–305.

2. YOUNG, B.J.J., R.K. MALLAYA, R.D.J LESLIE, et al. 1979. Anti-keratin antibodies inrheumatoid arthritis. Br. Med. J. 2: 97–99.

FIGURE 2. Anticitrullinated viral peptide antibodies in familial cases of RA. Histo-gram bars indicate the levels of anticitrullinated peptides antibodies; groups B to L representfamilies in which 2 or more siblings are affected by RA. In group A, a healthy sibling is alsotested (white bar). The affected siblings are, in most cases, both positive or both negativefor antipeptide antibodies.

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3. GIRBAL, E., M. SEBBAG, V. GOMES-DAUDRIX, et al. 1993. Characterisation of the ratoesophagus epithelium antigens defined by the so-called “antikeratin antibodies”,specific for rheumatoid arthritis. Ann. Rheum. Dis. 52: 749–757.

4. SIMON, M., E. GIRBAL, M. SEBBAG, et al. 1993. The cytokeratin filament-aggregatingprotein filaggrin is the target of so-called “antikeratin antibodies”, autoantibodiesspecific for rheumatoid arthritis. J. Clin. Invest. 92: 1387–1393.

5. SEBBAG, M., M. SIMON, C. VINCENT, et al. 1995. The antiperinuclear factor and the so-called antikeratin antibodies are the same rheumatoid arthritis–specific autoantibodies.J. Clin. Invest. 95: 2672–2679.

6. GIRBAL-NEUHAUSER, E., J.J. DURIEUX, M. ARNAUD, et al. 1999. The epitopes targeted bythe rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationallygenerated on various sites of (pro)filaggrin by deimination of arginine residues. J.Immunol. 162: 585–594.

7. SCHELLEKENS, G.A., B.A.W. DE JONG, F.H.J. VAN DEN HOOGEN, et al. 1998. Citrulline isan essential constituent of antigenic determinants recognized by rheumatoid arthritis–specific autoantibodies. J. Clin. Invest. 101: 273–281.

8. SCHELLEKENS, G.A., H. VISSER, B.A.W. DE JONG, et al. 2000. The diagnostic propertiesof rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. ArthritisRheum. 43: 155–163.

9. SABBATINI, A., S. BOMBARDIERI, P. MIGLIORINI. 1993. Autoantibodies from patientswith systemic lupus erythematosus bind a shared sequence of SmD and Epstein-Barrvirus-encoded nuclear antigen EBNA I. Eur. J. Immunol. 23: 1146–1152.

10. MARCHINI, B., M.P. DOLCHER, A. SABBATINI, et al. 1994. Immune response to differentsequences of the EBNA I molecule in Epstein-Barr virus-related disorders and inautoimmune diseases. J. Autoimmun. 7: 179–191.

11. ARNETT, F.C., S.M. EDWORTHY, D.A. BLOCH, et al. 1988. The American RheumatismAssociation 1987 revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum. 31: 315–322

12. TAN, E.M., A.S. COHEN, J.P. FRIES, et al. 1982. The 1982 revised criteria for the classi-fication of systemic lupus erythematosus. Arthritis Rheum. 25: 1271–1277.

13. SUBCOMMITTEE FOR SCLERODERMA CRITERIA OF THE AMERICAN RHEUMATISM ASSOCIATION

DIAGNOSTIC AND THERAPEUTIC CRITERIA COMMITTEE. 1980. Preliminary criteria forthe classification of systemic sclerosis (scleroderma). Arthritis Rheum. 23: 581–590.

14. PRATESI, R. et al. 2005. Forthcoming.15. ANZILOTTI, C. et al. 2005. Forthcoming.16. RADWAY-BRIGHT, E.L., C.T. RAVIRAJAN & D.A. ISENBERG. 2000. The prevalence of

antibodies to anionic phospholipids in patients with the primary antiphospholipidsyndrome, systemic lupus erythematosus and their relatives and spouses. Rheumatology39: 427–431.

17. FOLWACZNY, C., N. NOEHL, K. TSCHOP, et al. 1997. Goblet cell autoantibodies inpatients with inflammatory bowel disease and their first-degree relatives. Gastro-enterology 113: 101–106.

18. BRIX, T.H., P.S. HANSEN, K.O. KYVIK, et al. 2004. Aggregation of thyroid autoantibodiesin first-degree relatives of patients with autoimmune thyroid disease is mainly due togenes: a twin study. Clin. Endocrinol. (Oxf.) 60: 329–334.

19. HILL, J.A., S. SOUTHWOOD, A. SETTE, et al. 2003. Cutting edge: the conversion of arginineto citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis–associated HLA-DRB1*0401 MCH class II molecule. J. Immunol. 171: 538–541.

20. SUZUKI, A., R. YAMADA, X. CHANG, et al. 2003. Functional haplotypes of PADI4,encoding citrullinating enzyme peptidylarginine deiminase 4, are associated withrheumatoid arthritis. Nat. Genet. 34: 395–402.

21. BARTON, A., J. BOWES, S. EYRE, et al. 2004. Functional haplotype of the PADI4 geneassociated with rheumatoid arthritis in a Japanese population is not associated in aUnited Kingdom population. Arthritis Rheum. 50: 1117–1121.

22. CAPONI, L., E. PETIT-TEIXEIRA, M. SEBBAG, et al. 2005. A family-based study shows noassociation between rheumatoid arthritis and the PADI4 gene in a French Caucasianpopulation. Ann. Rheum. Dis. 64: 587–593.

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23. MASSON-BESSIER, C., M. SEBBAG, E. GIRBAL-NEUHAUSER, et al. 2001. The major synovialtarget of the rheumatoid arthritis–specific antifilaggrin autoantibodies are deiminatedforms of the alpha- and beta-chains of fibrin. J. Immunol. 166: 4177–4184.

24. VAN VENROOIJ, W.J. & G.J. PRUIJN. 2000. Citrullination: a small change for a proteinwith great consequences for rheumatoid arthritis. Arthritis Res. 2: 249–251.

25. SEBBAG, M., S. CHAPUY-REGAUD, I. AUGER, et al. 2004. Clinical and pathophysiologi-cal significance of the autoimmune response to citrullinated proteins in rheumatoidarthritis. Joint Bone Spine 71: 493–502.